Some of the information set forth herein has been published. See Pyle, A. M. and Barton, J. K., Mixed Ligand Complexes and Uses Thereof as Binding Agents to DNA, Inorganic Chemistry, 1987, 26:3820-3823, which was distributed by the published on Nov. 6, 1987.
Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
There has been considerable interest in elucidating those factors which determine affinity and selectivity in binding of small molecules to DNA. (66-72) A quantitative understanding of such factors which determine recognition of DNA sites would be valuable in the rational design of sequence-specific DNA binding molecules for application in chemotherapy and in the development of tools for biotechnology. Much work has focused on the elucidation of non-covalent interactions with DNA by small natural products and their synthetic derivatives. (67-72) These small molecules are stabilized in binding to DNA through a series of weak interactions, such as the .pi.-stacking interactions associated with intercalation of aromatic heterocyclic groups between the base pairs, and hydrogen bonding and Van der Waals interactions of functionalities bound along the groove of the DNA helix. It would be valuable to understand quantitively the contributions from these different modes to stabilization of the bound complex at a DNA site.
Previous work has focused on the examination of non-covalent interactions with DNA of transition metal complexes of phenanthroline. (66, 73-77) The cationic complexes has been found both to intercalate into DNA and to bind non-covalently in a surface-bound or gove-bound fashion. These interactions with DNA have been characterized largely through spectroscopic and photophysical studies, and determinations of enantiomeric selectivities associated with binding by the metal complexes have been helpful also in establishing models. (73, 74) On the basis of these investigations, intercalation likely occurs preferentially from the major groove of the DNA helix and is favored for the .DELTA. isomer into a right-handed helix. In the case of the surface-bound interaction, it likely occurs along the minor groove of the helix and it is the 1/3 isomer which is favored in surface-binding to right-handed DNA helices. FIG. 5 illustrates models for these binding interactions.
Based upon these binding interactions, derivatives of tris (phenanthroline) complexes have been developed which recognize selectively different conformations of DNA. By matching shapes and symmetries of the metal complexes to those of DNA conformations, probes for A-and Z-DNA have been designed. (75) Most recently, a diphenylphenanthroline complex of rhodium (III) has been found to induce double-stranded cleavage at cruciform sites upon photoactivation. (76). Although these complexes lack hydrogen bonding donors and acceptors and therefore must be associating with DNA only through a mixture of Van der Waals and intercalactive interactions, a high level of specificity is associated with the recognition of different DNA sites by these complexes.
The present invention involves mixed ligand complexes and complexes having three phenanthrenequinone diiamine ligands. The mixed ligand complexes of ruthenium (II) were explored for their interactions with B-DNA using a variety of biophysical and spectroscopic methods. Mixed ligand complexes of phenanthroline, phenanthrenequinonediimine, and derivatives thereof have been found to be useful for the construction and characterization of DNA-binding molecules. The ruthenium (II) complexes are particularly useful owing to their intense optical absorption and emission, their relative ease of preparation, and their inertness to substitution and racemization. (77-79).
The technique of DNA footprinting has been used extensively to observe the site-specific binding of proteins, peptides, and drugs to DNA (1-4). Using a variety of chemical and enzymatic footprinting agents, it has been possible to determine the relative binding site sizes and locations for hundreds of DNA-binding proteins. Subtle molecular interactions between DNA and transcription factor, repressor, and other constituents of the transcriptional apparatus are being actively explored using DNA footprinting (5).
Given the power of this methodology, extensive efforts to find new, high resolution footprinting reagents are underway. The most popular and the original footprinting reagent is DNase I, a large nuclease which cleaves with some preference for sequences of intermediate groove widths (6). This level of sequence-neutrality is sufficient for determining the binding sites of large DNA binding proteins. However, small peptides or proteins which bind to sequences insensitive to attack by DNase I can be difficult to visualize. Many chemical footprinting reagents such as Cu(phen).sub.2 + and metalloporphyrins share this inherent problem (7,8). In order to examine DNA binding interactions at higher resolution, many workers have turned to MPE-Fe(II), the first synthetic footprinting reagent and a remarkable tool with respect to its sequence neutrality. An intercalating dye tethered to an Fe(EDTA).sup.2- moiety, MPE-Fe(II) has been useful in elucidating the binding sites and sizes of small natural products as well as proteins (9-12). More recently, the clever application to footprinting of Fe(EDTA).sup.2- itself, without a tethered DNA-binding moiety, has been made (3,13). Both for Fe(EDTA).sup.2- and ME-Fe(II), cleavage results from the diffusion to the DNA helix of hydroxyl radicals, generated in the presence of peroxide and a reducing agent (3,9,13). FE(EDTA).sup.2-, which as an anionic species generates the radicals far from the DNA surface, also shows a high level of sequence neutrality, but since the radical generator does not bind to the DNA, high concentrations of reagents are required. Additionally a drawback with respect to both complexes has been their sensitivity to the presence of various common additives, such as glycerol or Mg.sup.2+, and their requirements for high concentrations of chemical activators.
Some techniques of photofootprinting have also been developed. An advantage of this method is that the activation of the DNA cleavage reaction is controlled by light, eliminating the need for adding other chemicals to the protein solution. These techniques include ultraviolet footprinting (14), photofootprinting in the presence of uranyl salts (15), and that in the presence of psoralen or its analogs (16). Ultraviolet light photofootprinting has been applied in vivo as well as in vitro. This technique requires chemical treatment after the photocleavage reaction, however, and the obtained results are sometimes complicated because of differential enhancements due to DNA-protein crosslinking. The second technique, using uranyl salts, shows excellent sequence neutrality but high concentrations of the uranyl salts are required, which may perturb the protein interactions with the DNA or the DNA structure itself. Psoralen footprinting lacks in sequence-neutrality. Owing to these difficulties, despite the inherent advantages of light activation, these photofootprinting reagents have not been widely applied.
Recently, coordinatively saturated phenanthrenequinone diimine complexes of rhodium (III) have been reported to cleave DNA efficiently upon irradiation with long-wavelength ultraviolet light (17). Photocleavage with Rh(phi).sub.2 (bpy).sup.3+ yields sharp, sequence-neutral cleavage of linear DNA fragments. The addition of free metal ions, chelators, or oxidizing agents is not necessary in this system because the Rh(phi).sub.2 (bpy).sup.3+ complex is fully assembled and requires activation only by light. The structure of Rh(phi).sub.2 (bpy).sup.3+ is schematically illustrated below. ##STR2##
Rh(phi).sub.2 (bpy).sup.3+ is a high-resolution photofootprinting reagent which successfully maps the precise binding locations and site sizes of distamycin-A and the restriction endonuclease EcoRI. This is the first report of a chemical footprint for EcoRI and the first example of a footprint which reflects the proper site size (18).
Rh(phi).sub.2 (bpy).sup.3+ is able to detect both EcoRI bound in the major groove of DNA and the small peptide distamycin, bound in the minor groove. Footprinting with Rh(phi).sub.2 (bpy).sup.3+ is not inhibited by moderate concentrations of salts, EDTA, glycerol, or reducing agents, many of which are sometimes necessary to obtain a native interaction of DNA with protein. The complex is ease to handle, being very stable under ordinary conditions and requiring no complicated reaction conditions. Activation with low energy light from a lamp or transilluminator permits excellent experimental control over Rh(phi).sub.2 (bpy).sup.3+ footprinting, an absolute requirement for application in vivo.
Extensive data has been accumulated on the luminescence properties of ruthenium (II) polypyridyls, the results of which in sum, suggest that the complexes are extremely useful luminescent labels for DNA. Recently, there has been concern with the use of these complexes as probes for specific sites of binding on the DNA helix. Among the reasons for their use in this work is that each complex can be resolved into stable isomers.sup.(34) and that their metal-to-ligand-charge-transfer (MLCT) excited states are easily accessible.sup.(35). It has previously been found that B-form DNA selectively bound the .DELTA.-enantiomers of Ru(phen).sub.3.sup.2+ and Ru(dip).sub.3.sup.2+ over the .OMEGA.-forms.sup.(36). More recently it was demonstrated that Z-form DNA selectively binds the .OMEGA.-enantiomers of Ru(phen).sub.3.sup.2+ and Ru(dip).sub.3.sup.2+(81). With this in mind, there is considerable interest in the development of new transition metal probes which would be more general in its binding and provide more information about its local DNA environment. Specifically, a complex whose luninescence properties would respond to subtle changes in environment upon binding.
The present invention involves, one such candidate, because of high binding ability to DNA is Ru(bpy)2(dppz).sup.2+ (bpy=2,2'-bipyridine, dppz=dipyrido[3,2: a-2',3'-cphenazine).sup.(38) which does not luminesce in aqueous solution. Ru(bpy).sub.2 (dppz).sup.2+ was found to have appreciable luminescence in ethanol ( .lambda..sub.irr =482 nm, .lambda..sub.max =610 nm) and in acetonitrile ( .lambda..sub.irr =482 nm, .lambda..sub.max =615 nm) The ancillary bpy ligands assures that intercalation only occurs via the dppz ligand. Furthermore, the two bpy ligands are not expected to provide enantiomeric selectivity for this complex.sup.(36). In addition the LUMO of this complex is described as having a very large electron density on the phenazine nitrogens.sup.(39), because this ligand is believed to extensively intercalate this provides an excellent probe of the helix environment. The major nonradiative deactivation pathway probably involves the protonation of the phenazine nitrogens in the excited state, which potentially provides an excellent probe of the interior of the DNA helix upon intercalation.